This proposal seeks to inactivate HIV by in vivo genetic modification. The development of a new class of therapeutics that can trigger rearrangements in genomic DNA is anticipated to have a significant impact on the treatment of HIV disease and AIDS-related malignancies. A technology has been developed for creating novel DNA-binding proteins from predefined zinc finger (ZnFn) modules. Such proteins can be rapidly assembled to recognize unique sites within the HIV genome with high affinity and specificity. In previous work, these proteins were used to create targeted artificial transcription factors. This proposal aims to create novel site-specific endonucleases by fusing the catalytic domains of naturally occurring enzymes to gene-specific ZnFn proteins. The reagents will be applied towards the long-term goal of engineering HIV-resistant hematopoetic stem cells. The endonuclease-based approach offers advantages over other gene regulation strategies because the modifications will be permanent and transmitted to progeny cells. Expression of the therapeutic gene will be required only transiently. The DNA form of the HIV genome represents a novel target for therapeutic intervention, and structural features of the genome make it ideally suited for the modifications described. The therapeutic potential of controlling HIV infection will be assessed in a novel cell culture model system. In principle, this approach could also be applied to inactivate HIV in latently infected reservoir cells. Currently no technology exists that can destroy the virus in this cell population. The net result of this study will be an increased understanding of inducing genomic modifications in cells, insights into the structure/function relationship of DNA modifying enzymes, and the development of a new class of molecular therapeutics that should find broad application in the study and treatment of HIV/AIDS and other diseases. The specific aims of this proposal are to: 1) Design and prepare polydactyl ZnFn proteins that will direct chimeric endonucleases to specific target sites in HIV-1 and viral co-receptors. 2) Produce targeted endonucleases by fusing known catalytic domains to the ZnFn DNA-binding proteins of Aim 1. Assess and optimize the ability of the chimeric enzymes to permanently inactivate a model retrovirus substrate. [unreadable] [unreadable] [unreadable]